Estrogen receptors (ER) alpha and beta are members of the nuclear receptor superfamily of proteins that regulate target gene transcription in response to hormones. Although co-expressed in certain tissues, the properties of ERalpha-ERbeta heterodimers are unknown. A number of studies indicate that ERbeta is a dominant negative inhibitor of ERalpha action, but these studies used an N-terminal truncated version of ERbeta and therefore further experiments are needed to address differences between the long and short forms of ERbeta. Tamoxifen (TAM) binds ER and is used to prevent recurrence of estrogen-dependent breast tumors and has mixed agonist-antagonist effects in other estrogen target tissues. The precise molecular mechanisms allowing estrogen response element (ERE) - containing genes to be selectively responsive to TAM versus estradiol are not well understood and may play a role in TAM resistant breast cancer. Several classes of nuclear accessory proteins are recruited to ERalpha and act as coactivator or corepressor complexes to activate or suppress transcription from EREs to which ERalpha is bound, usually in the promoter region. We reported significant differences in the ligand and ERE binding characteristics and transcriptional responsiveness of ERalpha and ERbeta, revealed by the use of natural and synthetic EREs that varied in sequence and spacing. Of note was our finding that the conformation of ERalpha changes when bound to different EREs, confirming our central hypothesis that DNA is an allosteric modulator of ER action. We hypothesize that these DNA-induced alterations in ER conformation will impact ER interaction with and activation by coactivator proteins. Comparatively little is known about ERbeta interaction with coactivators or corepressors. Using purified ERalpha and ERbeta for FRET and human normal and neoplastic mammary cells for transfection assays and ChIP, we will test the hypotheses that: ERalpha/ERbeta heterodimer differs in ERE and coactivator interaction from either ERalpha or ERbeta homodimers; alterations in ERalpha or ERbeta conformation induced by binding to different ERE sequences modulates ER interaction with coactivators SRC-1, SRC-2 (GRIP1), SRC-3 (AIB1), PRMT, and SRA; ERE regulated ER-coactivator interaction in turn modulates acetylation of histones H3 and H4, and the assembly of the coactivator/RNA polymerase II transcription initiation complex. Results will define the molecular mechanisms of ERalpha, ERbeta, and ERalpha/beta heterodimers as transcriptional regulators, with emphasis on the action of antiestrogens.